    Ahmad-Nejad, P., H. Hacker, M. Rutz, S. Bauer, R. M. Vabulas, and H. Wagner. 2002. Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur J Immunol 32:1958-68.    Aldovini, A., and R. A. Young. 1991. Humoral and cell-mediated immune responses to live recombinant BCG-HIV vaccines. Nature 351:479-82.    Andersson, G. E., and P. M. Sharp. 1996. Codon usage in the Mycobacterium tuberculosis complex. Microbiology 142:915-25.    Appay, V., D. F. Nixon, S. M. Donahoe, G. M. Gillespie, T. Dong, A. King, G. S. Ogg, H. M. Spiegel, C. Conlon, C. A. Spina, D. V. Havlir, D. D. Richman, A. Waters, P. Easterbrook, A. J. McMichael, and S. L. Rowland-Jones. 2000. HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function. J Exp Med 192:63-75.    Bange, F. C., F. M. Collins, and W. R. Jacobs, Jr. 1999. Survival of mice infected with Mycobacterium smegmatis containing large DNA fragments from Mycobacterium tuberculosis. Tuber Lung Dis 79:171-80.    Barouch, D. H., P. F. McKay, S. M. Sumida, S. Santra, S. S. Jackson, D. A. Gorgone, M. A. Lifton, B. K. Chakrabarti, L. Xu, G. J. Nabel, and N. L. Letvin. 2003. Plasmid chemokines and colony-stimulating factors enhance the immunogenicity of DNA priming-viral vector boosting human immunodeficiency virus type 1 vaccines. J Virol 77:8729-35.    Barouch, D. H., M. G. Pau, J. H. Custers, W. Koudstaal, S. Kostense, M. J. Havenga, D. M. Truitt, S. M. Sumida, M. G. Kishko, J. C. Arthur, B. Korioth-Schmitz, M. H. Newberg, D. A. Gorgone, M. A. Lifton, D. L. Panicali, G. J. Nabel, N. L. Letvin, and J. Goudsmit. 2004. Immunogenicity of recombinant adenovirus serotype 35 vaccine in the presence of pre-existing anti-Ad5 immunity. J Immunol 172:6290-7.    Beltan, E., L. Horgen, and N. Rastogi. 2000. Secretion of cytokines by human macrophages upon infection by pathogenic and non-pathogenic mycobacteria. Microb Pathog 28:313-8.    Calarota, S., G. Bratt, S, Nordlund, J. Hinkula, A. C. Leandersson, E. Sandstrom, and B. Wahren. 1998. Cellular cytotoxic response induced by DNA vaccination in HIV-1-infected patients. Lancet 351:1320-5.    Cheadle, E. J., D. O'Donnell, P. J. Selby, and A. M. Jackson. 2005. Closely related mycobacterial strains demonstrate contrasting levels of efficacy as antitumor vaccines and are processed for major histocompatibility complex class I presentation by multiple routes in dendritic cells. Infect Immun 73:784-94.    Connell, N. D., E. Medina-Acosta, W. R. McMaster, B. R. Bloom, and D. G. Russell. 1993. Effective immunization against cutaneous leishmaniasis with recombinant bacille Calmette-Guerin expressing the Leishmania surface proteinase gp63. Proc Natl Acad Sci USA 90:11473-7.    Converse, S. E., and J. S. Cox. 2005. A protein secretion pathway critical for Mycobacterium tuberculosis virulence is conserved and functional in Mycobacterium smegmatis. J Bacteriol 187:1238-45.    de Mendonca-Lima, L., M. Picardeau, C. Raynaud, J. Rauzier, Y. O. de la Salmoniere, L. Barker, F. Bigi, A. Cataldi, B. Gicquel, and J. M. Reyrat. 2001. Erp, an extracellular protein family specific to mycobacteria. Microbiology 147:2315-20.    de Miranda, A. B., F. Alvarez-Valin, K. Jabbari, W. M. Degrave, and G. Bernardi. 2000. Gene expression, amino acid conservation, and hydrophobicity are the main factors shaping codon preferences in Mycobacterium tuberculosis and Mycobacterium leprae. J Mol Evol 50:45-55.    Fennelly, G. J., J. L. Flynn, V. ter Meulen, U. G. Liebert, and B. R. Bloom. 1995. Recombinant bacille Calmette-Guerin priming against measles. J Infect Dis 172:698-705.    Fuller, M. J., and A. J. Zajac. 2003. Ablation of CD8 and CD4 T cell responses by high viral loads. J Immunol 170:477-86.    Gheorghiu, M., M. R. Lagranderie, B. M. Gicquel, and C. D. Leclerc. 1994. Mycobacterium bovis BCG priming induces a strong potentiation of the antibody response induced by recombinant BCG expressing a foreign antigen. Infect Immun 62:4287-95.    Grode, L., M. Kursar, J. Fensterle, S. H. Kaufmann, and J. Hess. 2002. Cell-mediated immunity induced by recombinant Mycobacterium bovis Bacille Calmette-Guerin strains against an intracellular bacterial pathogen: importance of antigen secretion or membrane-targeted antigen display as lipoprotein for vaccine efficacy. J Immunol 168:1869-76.    Hetzel, C., R. Janssen, S. J. Ely, N. M. Kristensen, K. Bunting, J. B. Cooper, J. R. Lamb, D. B. Young, and J. E. Thole. 1998. An epitope delivery system for use with recombinant mycobacteria. Infect Immun 66:3643-8.    Honda, M., K. Matsuo, T. Nakasone, Y. Okamoto, H. Yoshizaki, K. Kitamura, W. Sugiura, K. Watanabe, Y. Fukushima, S. Haga, Y. Katsura, H. Tasaka, K. Komuro, T. Yamada, T. Asano, A. Yamazaki, and S. Yamazaki. 1995. Protective immune responses induced by secretion of a chimeric soluble protein from a recombinant Mycobacterium bovis bacillus Calmette-Guerin vector candidate vaccine for human immunodeficiency virus type 1 in small animals. Proc Natl Acad Sci USA 92:10693-7.    Huebner, R. E. 1996. BCG vaccination in the control of tuberculosis. Curr Top Microbiol Immunol 215:263-82.    Huster, K. M., V. Busch, M. Schiemann, K. Linkemann, K. M. Kerksiek, H. Wagner, and D. H. Busch. 2004. Selective expression of IL-7 receptor on memory T cells identifies early CD40L-dependent generation of distinct CD8+ memory T cell subsets. Proc Natl Acad Sci USA 101:5610-5.    Kaech, S. M., and R. Ahmed. 2001. Memory CD8+ T cell differentiation: initial antigen encounter triggers a developmental program in naive cells. Nat Immunol 2:415-22.    Kamath, A. B., J. Woodworth, X. Xiong, C. Taylor, Y. Weng, and S. M. Behar. 2004. Cytolytic CD8+ T cells recognizing CFP10 are recruited to the lung after Mycobacterium tuberculosis infection. J Exp Med 200:1479-89.    Kaufmann, S. H., and U. E. Schaible. 2005. Antigen presentation and recognition in bacterial infections. Curr Opin Immunol 17:79-87.    Kuehnel, M. P., R. Goethe, A. Habermann, E. Mueller, M. Rohde, G. Griffiths, and P. Valentin-Weigand. 2001. Characterization of the intracellular survival of Mycobacterium avium ssp. paratuberculosis: phagosomal pH and fusogenicity in J774 macrophages compared with other mycobacteria. Cell Microbiol 3:551-66.    Kulkarni, H. R., and S. P. Zodpey. 1999. Differential protective effect of bacillus Calmette-Guerin vaccine against multibacillary and paucibacillary leprosy in Nagpur, India. Public Health 113:311-3.    Lagranderie, M., A. M. Balazuc, B. Gicquel, and M. Gheorghiu. 1997. Oral immunization with recombinant Mycobacterium bovis BCG simian immunodeficiency virus nef induces local and systemic cytotoxic T-lymphocyte responses in mice. J Virol 71:2303-9.    Langermann, S., S. R. Palaszynski, J. E. Burlein, S. Koenig, M. S. Hanson, D. E. Briles, and C. K. Stover. 1994. Protective humoral response against pneumococcal infection in mice elicited by recombinant bacille Calmette-Guerin vaccines expressing pneumococcal surface protein A. J Exp Med 180:2277-86.    Lee, M. H., L. Pascopella, W. R. Jacobs, Jr., and G. F. Hatfull. 1991. Site-specific integration of mycobacteriophage L5: integration-proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis, and bacille Calmette-Guerin. Proc Natl Acad Sci USA 88:3111-5.    Letvin, N. L. 2002. Strategies for an HIV vaccine. J Clin Invest 110:15-20.    Luo, Y., X. Chen, A. Szilvasi, and M. A. O'Donnell. 2000. Co-expression of interleukin-2 and green fluorescent protein reporter in mycobacteria: in vivo application for monitoring antimycobacterial immunity. Mol Immunol 37:527-36.    MacGregor, R. R., J. D. Boyer, K. E. Ugen, K. E. Lacy, S. J. Gluckman, M. L. Bagarazzi, M. A. Chattergoon, Y. Baine, T. J. Higgins, R. B. Ciccarelli, L. R. Coney, R. S. Ginsberg, and D. B. Weiner. 1998. First human trial of a DNA-based vaccine for treatment of human immunodeficiency virus type 1 infection: safety and host response. J Infect Dis 178:92-100.    Matsumoto, S., H. Yukitake, H. Kanbara, and T. Yamada. 1998. Recombinant Mycobacterium bovis bacillus Calmette-Guerin secreting merozoite surface protein 1 (MSP1) induces protection against rodent malaria parasite infection depending on MSP1-stimulated interferon gamma and parasite-specific antibodies. J Exp Med 188:845-54.    McKay, P. F., J. E. Schmitz, D. H. Barouch, M. J. Kuroda, M. A. Lifton, C. E. Nickerson, D. A. Gorgone, and N. L. Letvin. 2002. Vaccine protection against functional CTL abnormalities in simian human immunodeficiency virus-infected rhesus monkeys. J Immunol 168:332-7.    Mederle, I., I. Bourguin, D. Ensergueix, E. Badell, J. Moniz-Peireira, B. Gicquel, and N. Winter. 2002. Plasmidic versus insertional cloning of heterologous genes in Mycobacterium bovis BCG: impact on in vivo antigen persistence and immune responses. Infect Immun 70:303-14.    Molnar-Kimber, K. L., D. H. Sterman, M. Chang, E. H. Kang, M. ElBash, M. Lanuti, A. Elshami, K. Gelfand, J. M. Wilson, L. R. Kaiser, and S. M. Albelda. 1998. Impact of preexisting and induced humoral and cellular immune responses in an adenovirus-based gene therapy phase I clinical trial for localized mesothelioma. Hum Gene Ther 9:2121-33.    Nascimento, I. P., W. O. Dias, R. P. Mazzantini, E. N. Miyaji, M. Gamberini, W. Quintilio, V. C. Gebara, D. F. Cardoso, P. L. Ho, I. Raw, N. Winter, B. Gicquel, R. Rappuoli, and L. C. Leite. 2000. Recombinant Mycobacterium bovis BCG expressing pertussis toxin subunit S1 induces protection against an intracerebral challenge with live Bordetella pertussis in mice. Infect Immun 68:4877-83.    Newton, J. A., Jr., P. J. Weiss, W. A. Bowler, and E. C. Oldfield, 3rd. 1993. Soft-tissue infection due to Mycobacterium smegmatis: report of two cases. Clin Infect Dis 16:531-3.    Neyrolles, O., K. Gould, M. P. Gares, S. Brett, R. Janssen, P. O'Gaora, J. L. Herrmann, M. C. Prevost, E. Perret, J. E. Thole, and D. Young. 2001. Lipoprotein access to MHC class I presentation during infection of murine macrophages with live mycobacteria. J Immunol 166:447-57.    Noss, E. H., R. K. Pai, T. J. Sellati, J. D. Radolf, J. Belisle, D. T. Golenbock, W. H. Boom, and C. V. Harding. 2001. Toll-like receptor 2-dependent inhibition of macrophage class II MHC expression and antigen processing by 19-kDa lipoprotein of Mycobacterium tuberculosis. J Immunol 167:910-8.    Ozinsky, A., D. M. Underhill, J. D. Fontenot, A. M. Hajjar, K. D. Smith, C. B. Wilson, L. Schroeder, and A. Aderem. 2000. The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. Proc Natl Acad Sci USA 97:13766-71.    Pai, R. K., M. E. Pennini, A. A. Tobian, D. H. Canaday, W. H. Boom, and C. V. Harding. 2004. Prolonged toll-like receptor signaling by Mycobacterium tuberculosis and its 19-kilodalton lipoprotein inhibits gamma interferon-induced regulation of selected genes in macrophages. Infect Immun 72:6603-14.    Pan, A., C. Dutta, and J. Das. 1998. Codon usage in highly expressed genes of Haemophillus influenzae and Mycobacterium tuberculosis: translational selection versus mutational bias. Gene 215:405-13.    Panina-Bordignon P, Tan A, Termijtelen A, Demotz S, Corradin G, Lanzavecchia A. 1989. Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells. Eur J Immunol 19:2237-42.    Pierre-Audigier, C., E. Jouanguy, S. Lamhamedi, F. Altare, J. Rauzier, V. Vincent, D. Canioni, J. F. Emile, A. Fischer, S. Blanche, J. L. Gaillard, and J. L. Casanova. 1997. Fatal disseminated Mycobacterium smegmatis infection in a child with inherited interferon gamma receptor deficiency. Clin Infect Dis 24:982-4.    Seaman, M. S., F. W. Peyerl, S. S. Jackson, M. A. Lifton, D. A. Gorgone, J. E. Schmitz, and N. L. Letvin. 2004. Subsets of memory cytotoxic T lymphocytes elicited by vaccination influence the efficiency of secondary expansion in vivo. J Virol 78:206-15.    Snapper, S. B., L. Lugosi, A. Jekkel, R. E. Melton, T. Kieser, B. R. Bloom, and W. R. Jacobs, Jr. 1988. Lysogeny and transformation in mycobacteria: stable expression of foreign genes. Proc Natl Acad Sci USA 85:6987-91.    Snapper, S. B., R. E. Melton, S. Mustafa, T. Kieser, and W. R. Jacobs, Jr. 1990. Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol 4:1911-9.    Staats, H. F., C. P. Bradney, W. M. Gwinn, S. S. Jackson, G. D. Sempowski, H. X. Liao, N. L. Letvin, and B. F. Haynes. 2001. Cytokine requirements for induction of systemic and mucosal CTL after nasal immunization. J Immunol 167:5386-94.    Stover, C. K., G. P. Bansal, M. S. Hanson, J. E. Burlein, S. R. Palaszynski, J. F. Young, S. Koenig, D. B. Young, A. Sadziene, and A. G. Barbour. 1993. Protective immunity elicited by recombinant bacille Calmette-Guerin (BCG) expressing outer surface protein A (OspA) lipoprotein: a candidate Lyme disease vaccine. J Exp Med 178:197-209.    Stover, C. K., V. F. de la Cruz, T. R. Fuerst, J. E. Burlein, L. A. Benson, L. T. Bennett, G. P. Bansal, J. F. Young, M. H. Lee, G. F. Hatfull, and et al. 1991. New use of BCG for recombinant vaccines. Nature 351:456-60.    Takahashi, H., Y. Nakagawa, C. D. Pendleton, R. A. Houghten, K. Yokomuro, R. N. Germain, and J. A. Berzofsky. 1992. Induction of broadly cross-reactive cytotoxic T cells recognizing an HIV-1 envelope determinant. Science 255:333-6.    Tobian, A. A., N. S. Potter, L. Ramachandra, R. K. Pai, M. Convery, W. H. Boom, and C. V. Harding. 2003. Alternate class I MHC antigen processing is inhibited by Toll-like receptor signaling pathogen-associated molecular patterns: Mycobacterium tuberculosis 19-kDa lipoprotein, CpG DNA, and lipopolysaccharide. J Immunol 171:1413-22.    Underhill, D. M., A. Ozinsky, A. M. Hajjar, A. Stevens, C. B. Wilson, M. Bassetti, and A. Aderem. 1999. The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens. Nature 401:811-5.    Via, L. E., D. Deretic, R. J. Ulmer, N. S. Hibler, L. A. Huber, and V. Deretic. 1997. Arrest of mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by rab5 and rab7. J Biol Chem 272:13326-31.    Via, L. E., R. A. Fratti, M. McFalone, E. Pagan-Ramos, D. Deretic, and V. Deretic. 1998. Effects of cytokines on mycobacterial phagosome maturation. J Cell Sci 111 (Pt 7): 897-905.    Wang, R., D. L. Doolan, T. P. Le, R. C. Hedstrom, K. M. Coonan, Y. Charoenvit, T. R. Jones, P. Hobart, M. Margalith, J. Ng, W. R. Weiss, M. Sedegah, C. de Taisne, J. A. Norman, and S. L. Hoffman. 1998. Induction of antigen-specific cytotoxic T lymphocytes in humans by a malaria DNA vaccine. Science 282:476-80.    Wei, J., J. L. Dahl, J. W. Moulder, E. A. Roberts, P. O'Gaora, D. B. Young, and R. L. Friedman. 2000. Identification of a Mycobacterium tuberculosis gene that enhances mycobacterial survival in macrophages. J Bacteriol 182:377-84.    Weiss, B. G., and S. Schlesinger. 1991. Recombination between Sindbis virus RNAs. J Virol 65:4017-25.    Wherry, E. J., and R. Ahmed. 2004. Memory CD8 T-cell differentiation during viral infection. J Virol 78:5535-45.    Wherry, E. J., J. N. Blattman, K. Murali-Krishna, R. van der Most, and R. Ahmed. 2003. Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment. J Virol 77:4911-27.    Wherry, E. J., V. Teichgraber, T. C. Becker, D. Masopust, S. M. Kaech, R. Antia, U. H. von Andrian, and R. Ahmed. 2003. Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat Immunol 4:225-34.    Wyatt, R., P. D. Kwong, E. Desjardins, R. W. Sweet, J. Robinson, W. A. Hendrickson, and J. G. Sodroski. 1998. The antigenic structure of the HIV gp120 envelope glycoprotein. Nature 393:705-11.    Yadav, M., S. K. Roach, and J. S. Schorey. 2004. Increased mitogen-activated protein kinase activity and TNF-alpha production associated with Mycobacterium smegmatis-but not Mycobacterium avium-infected macrophages requires prolonged stimulation of the calmodulin/calmodulin kinase and cyclic AMP/protein kinase A pathways. J Immunol 172:5588-97.    Yasutomi, Y., S. Koenig, S. S. Haun, C. K. Stover, R. K. Jackson, P. Conard, A. J. Conley, E. A. Emini, T. R. Fuerst, and N. L. Letvin. 1993. Immunization with recombinant BCG-SIV elicits SIV-specific cytotoxic T lymphocytes in rhesus monkeys. J Immunol 150:3101-7.    Young, D. B., and T. R. Garbe. 1991. Lipoprotein antigens of Mycobacterium tuberculosis. Res Microbiol 142:55-65.    Young, S. L., M. Murphy, X. W. Zhu, P. Harnden, M. A. O'Donnell, K. James, P. M. Patel, P. J. Selby, and A. M. Jackson. 2004. Cytokine-modified Mycobacterium smegmatis as a novel anticancer immunotherapy. Int J Cancer 112:653-60.    Zajac, A. J., J. N. Blattman, K. Murali-Krishna, D. J. Sourdive, M. Suresh, J. D. Altman, and R. Ahmed. 1998. Viral immune evasion due to persistence of activated T cells without effector function. J Exp Med 188:2205-13.
There remains an urgent need for an effective malaria vaccine. Based on previous studies demonstrating that immunization with Msp-1 protein can protect Aotus monkeys from severe forms of malaria, the demonstration that Msp-1 can be highly expressed in BCG and elicit antibody bodes well for a recombinant BCG vaccine to minimally prime for an effective malarial response.
There is also an urgent need for an HIV vaccine. Recombinant attenuated non-pathogenic mycobacteria expressing HIV immunogens are attractive vaccine candidates because of the proven safety and immunogenicity of Mycobacterium bovis BCG in humans as a vaccine against tuberculosis.
An effective HIV/AIDS vaccine will likely need to elicit virus-specific neutralizing antibodies and cytotoxic T lymphocyte (CTL) responses. Although an immunogen that induces antibodies that neutralize a diversity of primary HIV-1 isolates has not yet been defined, a number of strategies are being developed for generating HIV-1-specific CTL (Letvin, 2002). However, there are problems associated with each of these approaches for eliciting CTL that will likely limit their ultimate effectiveness. Plasmid DNA has not proven nearly as immunogenic in humans as it has in laboratory animals (Calarota et al., 1998; MacGregor et al., 1998; Wang et al., 1998). The immunogenicity of replication-defective adenovirus serotype 5 is limited in human populations by pre-existing serotype-specific anti-adenovirus antibodies (Molnar-Kimber et al., 1998). Pox-vectored vaccines only elicit very short-lived immunity in humans (Andrew McMichael, personal communication) and production problems have slowed the development of alphavirus-based vaccine vectors (Weiss and Schlesinger et al., 1991). Better vector systems will therefore be needed to induce anti-HIV-1 cellular immunity and prime for broadly neutralizing antibody responses.
Mycobacteria have features that make them attractive as potential HIV-1 vaccine vectors. They can be readily engineered to stably express transgenes and can elicit long-lasting cellular and mucosal immune responses (Lagranderie et al., 1997; Mederle et al., 2002). Most importantly, they have been used successfully as vaccines. The attenuated, non-pathogenic Mycobacterium bovis BCG is widely used as a vaccine for tuberculosis (TB) and leprosy (Huebner, 1996; Kulkarni and Zodpey, 1999). Recombinant BCG (rBCG) vaccine constructs have shown immunogenicity and protection in murine models against various infectious agents, including Borrelia burgdorferi, Streptococcus pneumoniae, Bordetella pertussis, rodent malaria, leishmania, and measles virus (Connell et al., 1993; Fennelly et al., 1995; Langermann et al., 1994; Matsumoto et al., 1998; Nascimento et al., 2000; Stover et al., 1993). In murine and monkey studies, we and others have shown that rBCG-elicited antibody and cell-mediated responses against HIV-1 and SIV antigens (Almad-Nejad et al., 2002; Honda et al., 1995; Stover et al., 1991; Yasutomi et al., 1993).
Mycobacterium smegmatis has a number of properties that may make it an effective vaccine vector. Some M. smegmatis strains are non-pathogenic and commensal in humans (Bange et al., 1999; Newton et al., 1993; Pierre-Audigier et al., 1997). Unlike other mycobacterial species such as BCG that survive in host cells for months by inhibiting phagosome maturation, M. smegmatis is rapidly destroyed by phagolysosomal proteases in the phagosomes of infected cells (Kuchnel et al., 2001; Luo et al., 2000; Via et al., 1997; 1998). Nevertheless, M. smegmatis can induce cytokine production by macrophages better than pathogenic mycobacterial species (Beltan et al., 2000; Yadev et al., 2004), and can activate and induce the maturation of dendritic cells better than BCG by upregulation of MHC I and costimulatory molecules (Cheadle et al., 2005). M. smegmatis can also access the MHC class I pathway for presentation of mycobacterial antigens more efficiently than BCG (Neyrolles et al., 2001). The present studies were initiated to assess the ability of recombinant M. smegmatis to elicit HIV-1 envelope-specific CD8+ T cell responses.
Since malaria & HIV coexist in developing and underdeveloped countries, there is a need for inexpensive vaccines for either pathogen. There is also a need for more effective malaria and HIV vaccines. The present invention addresses those needs.